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. 2021 Jan 27;9(2):259.
doi: 10.3390/microorganisms9020259.

Safety, Immunogenicity, and Protective Efficacy of a Chimeric A/B Live Attenuated Influenza Vaccine in a Mouse Model

Ekaterina Stepanova  1 , Elena Krutikova  1 , Pei-Fong Wong  1 , Victoria Matyushenko  1 , Ekaterina Bazhenova  1 , Irina Isakova-Sivak  1 , Larisa Rudenko  1
Affiliations

Safety, Immunogenicity, and Protective Efficacy of a Chimeric A/B Live Attenuated Influenza Vaccine in a Mouse Model

Ekaterina Stepanova et al. Microorganisms. .

Abstract

Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin-Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44+CD62L-) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP366); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.

Keywords: LAIV; chimeric hemagglutinin; genome packaging; influenza B virus; influenza virus; live attenuated influenza vaccine; reassortment; universal influenza vaccine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Experiment design scheme: the mice were twice immunized with experimental chimeric (H2B) vaccine or control live attenuated influenza vaccines (LAIVs). On day 3 after immunization, vaccine virus titers in the organs were assessed in 4 animals from each group. Three weeks after the second immunization, the animals were challenged with influenza A or B virus. Five animals stayed naïve for negative controls. Viral loads in the lungs were studied on day 3 (for influenza B) and day 5 (for influenza A) after the challenge. The number of animals in each group is indicated in frames.
Figure 2
Figure 2
Chimeric A/B genome composition and a scheme of hemagglutinin (HA) and neuraminidase (NA) genetic segments: the chimeric H2B reassortant virus contains six genetic segments from A/Leningrad/134/17/57 (A/Len/17) (H2N2) LAIV master donor virus (MDV) (shown in green). Segments encoding HA and NA are engineered constructs on the base of parts of A/Len/17 and B/Brisbane/60/2008 (B/Brisbane) (shown in red). In the scheme of segment constructions, exact numbers of nucleotides are specified. For the B/Brisbane HA ectodomain, the positions of start and end nucleotides in the B/Brisbane/60/08 HA segment are indicated. At the end of the B/HA ectodomain, His545 was changed to Tyr, corresponding to A/Len/17 appropriate position (shown with grey arrow). SP: HA signal peptide, TMD: transmembrane domain, CPD: cytoplasmic domain, UTR: untranslated region.
Figure 3
Figure 3
H2B chimera, A/Len/17, and B/Brisbane/60/08 growth kinetics in Madin–Darby Canine Kidney (MDCK) cells: viral replication was detected both by cell-based ELISA with monoclonal antibody to influenza nucleoprotein (NP) and by the HA assay of a cell-culture medium with 1% chicken red blood cells (RBC). (A) A/Len/17; (B) H2B chimeric LAIV; and (C) B/Brisbane/60/2008.
Figure 4
Figure 4
Mapping of the Gly 141 position on the B/Brisbane/60/2008 HA structure. Left: HA trimer, monomers are shown by different colors. Right: the receptor binding site area. The black arrow points to the 141 position. The figure was prepared on the base of PDB 4fqm [42] using Chimera 1.14 software.
Figure 5
Figure 5
Titers of the studied LAIV viruses in lungs and nasal turbinates (NTs) of C57BL/6J mice on day 3 after immunization (mean ± SD): (A) titers in nasal turbinates on day 3 post-immunization and (B) titers in lungs at day 3 post-immunization.
Figure 6
Figure 6
Serum antibody responses after immunization with the LAIVs studied: serum antibody titers were assessed 3 weeks after 2nd immunization. Titers were studied with 3 antigens (influenza B: B/Brisbane/60/2008; H2B LAIV; and influenza A: A/17/New York/2015/5364) in (A) hemagglutination inhibition (HAI) and (B) ELISA tests. Significant differences are indicated by dashes, p values are specified: HAI–Kruskal–Wallis ANOVA (Dunn’s multiple comparisons test); ELISA–ANOVA (Tukey’s multiple comparisons test).
Figure 7
Figure 7
Sera neutralizing activity after immunization with LAIVs studied. Neutralizing antibody titers were assessed in MNT 3 weeks after 2nd immunization, with 3 viruses: (A) B/Brisbane/60/2008; (B) H2B chimeric LAIV; (C) A/17/New York/2015/5364. Significant differences are indicated (Kruskal–Wallis ANOVA, Dunn’s multiple comparisons test).
Figure 8
Figure 8
Levels (%) of IFN-γ-secreting CD8+ cells and TEM cells (CD8+CD44+CD62L− phenotype) in spleens of immunized and control mice: splenocytes were isolated 5 days after challenge with influenza A virus, and T-cells were stimulated with influenza A NP366-374 CTL peptide (ASNENMDTM). (A) Representative flow cytometry plots of the IFNγ-secreting CD8+ effector memory T cells for each test group (shown in boxes). The numbers indicate the % of IFNγ-secreting cells among total effector memory CD8+ T cells., (B) levels of IFNγ-secreting CD8+ T cells, and (C) levels of IFNγ-secreting CD8+ TEM cells (CD8+CD44+CD62L− phenotype): significant differences are indicated on the graph (Kruskal–Wallis test, multiple comparisons with Dunn’s test); *** p < 0.0005; ** p < 0.005; and * p < 0.05.
Figure 9
Figure 9
Influenza B challenge virus (B/Brisbane/60/08) titers in lungs and nasal turbinates on 3 dpi: Animals were intranasally (i.n.) inoculated with challenge virus on day 42. Virus titers were studied at 3 dpi by titration in chicken embryos. Titer data are presented as mean ± SD. The p values are indicated on the graph (ANOVA, multiple comparisons with Dunnett’s test).
Figure 10
Figure 10
Influenza A challenge virus (A/California/07/2009 mouse-adapted strain) titers in lungs and nasal turbinates on 5 dpi: animals were i.n. inoculated with challenge virus on day 42. Virus titers were studied at 5 dpi by titration in chicken embryos. Titer data are presented as mean ± SD. The p values are indicated (ANOVA, multiple comparisons with Dunnett’s test).
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